T-type Ca2+ channels, SK2 channels and SERCAs gate sleep-related oscillations in thalamic dendrites.

T-type Ca2+ channels (T channels) underlie rhythmic burst discharges during neuronal oscillations that are typical during sleep. However, the Ca2+-dependent effectors that are selectively regulated by T currents remain unknown. We found that, in dendrites of nucleus reticularis thalami (nRt), intracellular Ca2+ concentration increases were dominated by Ca2+ influx through T channels and shaped rhythmic bursting via competition between Ca2+-dependent small-conductance (SK)-type K+ channels and Ca2+ uptake pumps. Oscillatory bursting was initiated via selective activation of dendritically located SK2 channels, whereas Ca2+ sequestration by sarco/endoplasmic reticulum Ca2+-ATPases (SERCAs) and cumulative T channel inactivation dampened oscillations. Sk2-/- (also known as Kcnn2) mice lacked cellular oscillations, showed a greater than threefold reduction in low-frequency rhythms in the electroencephalogram of non-rapid-eye-movement sleep and had disrupted sleep. Thus, the interplay of T channels, SK2 channels and SERCAs in nRt dendrites comprises a specialized Ca2+ signaling triad to regulate oscillatory dynamics related to sleep.

Nuclear calcium signaling: a cell within a cell

Calcium (Ca2+) is a versatile second messenger that regulates a wide range of cellular functions. Although it is not established how a single second messenger coordinates diverse effects within a cell, there is increasing evidence that the spatial patterns of Ca2+ signals may determine their specificity. Ca2+ signaling patterns can vary in different regions of the cell and Ca2+ signals in nuclear and cytoplasmic compartments have been reported to occur independently. No general paradigm has been established yet to explain whether, how, or when Ca2+ signals are initiated within the nucleus or their function. Here we highlight that receptor tyrosine kinases rapidly translocate to the nucleus. Ca2+ signals that are induced by growth factors result from phosphatidylinositol 4,5-bisphosphate hydrolysis and inositol 1,4,5-trisphosphate formation within the nucleus rather than within the cytoplasm. This novel signaling mechanism may be responsible for growth factor effects on cell proliferation.

Mechanism of Inducible Costimulator (ICOS)-mediated calcium signaling

Le Costimulateur Inductible (ICOS) est un r��cepteur exprim�� �� la surface des cellules T CD4 auxiliaires et T CD8 cytotoxiques. Il fut d��montr�� �� l���aide de mod��les murins de transplantation de moelle osseuse que ICOS joue un r��le important dans l���induction de la maladie du greffon contre l���h��te aigu��e (GVHD). ICOS potentialise deux signaux m��di��s par le r��cepteur de cellules T (TCR) : l���activation de la phosphoinositide 3-kinase (PI3K) ainsi que la mobilisation interne de calcium. En conditions in vitro, dans les cellules CD4 et CD8, ICOS r��ussi �� potentialiser le flux de calcium m��di�� par le TCR ind��pendamment de PI3K. La voie de signalisation de ICOS impliqu��e dans la GVHD demeure inconnue. Cependant, en utilisant une lign��e de souris ���knock-in��� nomm��e ICOS-Y181F, dans laquelle le cellules T ont s��lectivement perdu la capacit�� d���activer PI3K par l���entremise d���ICOS, nous avons d��montr�� que les cellules T peuvent utiliser un m��canisme ICOS ind��pendant de PI3K afin d���induire la GVHD. La mobilisation interne du Ca2+ m��ne �� l���activation de NFAT, un facteur de transcription cl�� r��gulant des g��nes comme IFN-��, qui exprime une des cytokines cl��s impliqu��es dans la GVHD. Nous ��mettons comme hypoth��se que la capacit�� pathog��nique intacte des cellules T ICOSY181F �� induire la GVHD, repose sur la signalisation du Ca2+ ind��pendante de PI3K. Le but de mon projet est d���identifier les r��sidus responsables de cette signalisation de Ca2+ m��di��e par ICOS ainsi que le m��canisme par lequel ce r��cepteur fonctionne. �� l���aide de la mutag��n��se dirig��e, j���ai g��n��r�� des mutants d���ICOS et j���ai analys�� par cytom��trie en flux leur capacit�� �� activer le flux de Ca2+. J���ai ainsi identifi�� un groupe de lysine sur la queue cytoplasmique d���ICOS situ�� �� proximit�� de la membrane comme ��tant essentiel �� la fonction de potentialisation du flux de Ca2+. Je fournis ��galement des preuves de l���implication de la kinase Lck, membre de la famille de kinases Src, dans la voie de signalisation de ICOS m��diant la potentialisation du flux de Ca2+. Ainsi, ICOS s���associe �� Lck et m��ne �� une augmentation de l���activation de PLC��1, la prot��ine effectrice cl�� causant la sortie de Ca2+ de la r��serve intracellulaire. En conclusion, notre ��tude permet de comprendre davantage une des voies de signalisation d���ICOS. L���influx de Ca2+ dans les cellules T implique la voie ICOS-Lck-PLC��1. Une compr��hension plus approfondie de cette voie de signalisation pourrait s���av��rer b��n��fique afin d�����laborer de nouvelles strat��gies menant �� la pr��vention de maladies reli��es �� ICOS, comme la GVHD.

Mechanism of acetylcholine-induced calcium signaling during neuronal differentiation of P19 embryonal carcinoma cells in vitro

Muscarinic (mAChRs) and nicotinic acetylcholine receptors (nAChRs) are involved in various physiological processes, including neuronal development. We provide evidence for expression of functional nicotinic and muscarinic receptors during differentiation of P19 carcinoma embryonic cells, as an in vitro model of early neurogenesis. We have detected expression and activity alpha(2)-alpha(7), beta(2), beta(4) nAChR and M1-M5 mAChR subtypes during neuronal differentiation. Nicotinic alpha(3) and beta(2) mRNA transcription was induced by addition of retinoic acid to P19 cells. Gene expression Of alpha(2), alpha(4)-alpha(7), beta(4) nAChR subunits decreased during initial differentiation and increased again when P19 cells underwent final maturation. Receptor response in terms of nicotinic agonist-evoked Ca2+, flux was observed in embryonic and neuronal-differentiated cells. Muscarinic receptor response, merely present in undifferentiated P19 cells, increased during neuronal differentiation. The nAChR-induced elevation of intracellular calcium ([Ca2+](i)) response in undifferentiated cells was due to Ca2+ influx. In differentiated P19 neurons the nAChR-induced [Ca2+](i) response was reduced following pretreatment with ryanodine, while the mAChR-induced response was unaffected indicating the contribution of Ca2+ release from ryanodine-sensitive stores to nAChR- but not mAChR-mediated Ca2+ responses. The presence of functional nAChRs in embryonic cells suggests that these receptors are involved in triggering Ca2+ waves during initial neuronal differentiation. (C) 2007 Elsevier Ltd. All rights reserved.

Functional and Structural Adaptations in the Pancreatic alpha-Cell and Changes in Glucagon Signaling During Protein Malnutrition

Funda����o de Amparo �� Pesquisa do Estado de S��o Paulo (FAPESP)

Ca2+-dependent repair of pneumolysin pores: A new paradigm for host cellular defense against bacterial pore-forming toxins

Pneumolysin (PLY), a key virulence factor of Streptococcus pneumoniae, permeabilizes eukaryotic cells by forming large trans-membrane pores. PLY imposes a puzzling multitude of diverse, often mutually excluding actions on eukaryotic cells. Whereas cytotoxicity of PLY can be directly attributed to the pore-mediated effects, mechanisms that are responsible for the PLY-induced activation of host cells are poorly understood. We show that PLY pores can be repaired and thereby PLY-induced cell death can be prevented. Pore-induced Ca2+ entry from the extracellular milieu is of paramount importance for the initiation of plasmalemmal repair. Nevertheless, active Ca2+ sequestration that prevents excessive Ca2+ elevation during the execution phase of plasmalemmal repair is of no less importance. The efficacy of plasmalemmal repair does not only define the fate of targeted cells but also intensity, duration and repetitiveness of PLY-induced Ca2+ signals in cells that were able to survive after PLY attack. Intracellular Ca2+ dynamics evoked by the combined action of pore formation and their elimination mimic the pattern of receptor-mediated Ca2+ signaling, which is responsible for the activation of host immune responses. Therefore, we postulate that plasmalemmal repair of PLY pores might provoke cellular responses that are similar to those currently ascribed to the receptor-mediated PLY effects. Our data provide new insights into the understanding of the complexity of cellular non-immune defense responses to a major pneumococcal toxin that plays a critical role in the establishment and the progression of life-threatening diseases. Therapies boosting plasmalemmal repair of host cells and their metabolic fitness might prove beneficial for the treatment of pneumococcal infections.

Oxidative Stress and Ca2+ Release Events in Mouse Cardiomyocytes

Cellular oxidative stress, associated with a variety of common cardiac diseases, is well recognized to affect the function of several key proteins involved in Ca2+ signaling and excitation-contraction coupling, which are known to be exquisitely sensitive to reactive oxygen species. These include the Ca2+ release channels of the sarcoplasmic reticulum (ryanodine receptors or RyR2s) and the Ca2+/calmodulin-dependent protein kinase II (CaMKII). Oxidation of RyR2s was found to increase the open probability of the channel, whereas CaMKII can be activated independent of Ca2+ through oxidation. Here, we investigated how oxidative stress affects RyR2 function and SR Ca2+ signaling in situ, by analyzing Ca2+ sparks in permeabilized mouse cardiomyocytes under a broad range of oxidative conditions. The results show that with increasing oxidative stress Ca2+ spark duration is prolonged. In addition, long and very long-lasting (up to hundreds of milliseconds) localized Ca2+ release events started to appear, eventually leading to sarcoplasmic reticulum (SR) Ca2+ depletion. These changes of release duration could be prevented by the CaMKII inhibitor KN93 and did not occur in mice lacking the CaMKII-specific S2814 phosphorylation site on RyR2. The appearance of long-lasting Ca2+ release events was paralleled by an increase of RyR2 oxidation, but also by RyR-S2814 phosphorylation, and by CaMKII oxidation. Our results suggest that in a strongly oxidative environment oxidation-dependent activation of CaMKII leads to RyR2 phosphorylation and thereby contributes to the massive prolongation of SR Ca2+ release events.

Reactive oxygen species and nitric oxide mediate plasticity of neuronal calcium signaling

Reactive oxygen species (ROS) and nitric oxide (NO) are important participants in signal transduction that could provide the cellular basis for activity-dependent regulation of neuronal excitability. In young rat cortical brain slices and undifferentiated PC12 cells, paired application of depolarization/agonist stimulation and oxidation induces long-lasting potentiation of subsequent Ca2+ signaling that is reversed by hypoxia. This potentiation critically depends on NO production and involves cellular ROS utilization. The ability to develop the Ca2+ signal potentiation is regulated by the developmental stage of nerve tissue, decreasing markedly in adult rat cortical neurons and differentiated PC12 cells.

Mitochondrial control of calcium-channel gating: A mechanism for sustained signaling and transcriptional activation in T lymphocytes

In addition to their well-known functions in cellular energy transduction, mitochondria play an important role in modulating the amplitude and time course of intracellular Ca2+ signals. In many cells, mitochondria act as Ca2+ buffers by taking up and releasing Ca2+, but this simple buffering action by itself often cannot explain the organelle's effects on Ca2+ signaling dynamics. Here we describe the functional interaction of mitochondria with store-operated Ca2+ channels in T lymphocytes as a mechanism of mitochondrial Ca2+ signaling. In Jurkat T cells with functional mitochondria, prolonged depletion of Ca2+ stores causes sustained activation of the store-operated Ca2+ current, ICRAC (CRAC, Ca2+ release-activated Ca2+). Inhibition of mitochondrial Ca2+ uptake by compounds that dissipate the intramitochondrial potential unmasks Ca2+-dependent inactivation of ICRAC. Thus, functional mitochondria are required to maintain CRAC-channel activity, most likely by preventing local Ca2+ accumulation near sites that govern channel inactivation. In cells stimulated through the T-cell antigen receptor, acute blockade of mitochondrial Ca2+ uptake inhibits the nuclear translocation of the transcription factor NFAT in parallel with CRAC channel activity and [Ca2+]i elevation, indicating a functional link between mitochondrial regulation of ICRAC and T-cell activation. These results demonstrate a role for mitochondria in controlling Ca2+ channel activity and signal transmission from the plasma membrane to the nucleus.

Two-dimensional confocal images of organization, density, and gating of focal Ca2+ release sites in rat cardiac myocytes

In cardiac myocytes Ca2+ cross-signaling between Ca2+ channels and ryanodine receptors takes place by exchange of Ca2+ signals in microdomains surrounding dyadic junctions, allowing first the activation and then the inactivation of the two Ca2+-transporting proteins. To explore the details of Ca2+ signaling between the two sets of receptors we measured the two-dimensional cellular distribution of Ca2+ at 240 Hz by using a novel confocal imaging technique. Ca2+ channel-triggered Ca2+ transients could be resolved into dynamic ���Ca2+ stripes��� composed of hundreds of discrete focal Ca2+ releases, appearing as bright fluorescence spots (radius ��� 0.5 ��m) at reproducible sites, which often coincided with t-tubules as visualized with fluorescent staining of the cell membrane. Focal Ca2+ releases triggered stochastically by Ca2+ current (ICa) changed little in duration (���7 ms) and size (���100,000 Ca ions) between ���40 and +60 mV, but their frequency of activation and first latency mirrored the kinetics and voltage dependence of ICa. The resolution of 0.95 �� 0.13 reproducible focal Ca2+ release sites per ��m3 in highly Ca2+-buffered cells, where diffusion of Ca2+ is limited to 50 nm, suggests the presence of about one independent, functional Ca2+ release site per half sarcomere. The density and distribution of Ca2+ release sites suggest they correspond to dyadic junctions. The abrupt onset and termination of focal Ca2+ releases indicate that the cluster of ryanodine receptors in individual dyadic junctions may operate in a coordinated fashion.

Competitive regulation of CaT-like-mediated Ca2+ entry by protein kinase C and calmodulin

A finely tuned Ca2+ signaling system is essential for cells to transduce extracellular stimuli, to regulate growth, and to differentiate. We have recently cloned CaT-like (CaT-L), a highly selective Ca2+ channel closely related to the epithelial calcium channels (ECaC) and the calcium transport protein CaT1. CaT-L is expressed in selected exocrine tissues, and its expression also strikingly correlates with the malignancy of prostate cancer. The expression pattern and selective Ca2+ permeation properties suggest an important function in Ca2+ uptake and a role in tumor progression, but not much is known about the regulation of this subfamily of ion channels. We now demonstrate a biochemical and functional mechanism by which cells can control CaT-L activity. CaT-L is regulated by means of a unique calmodulin binding site, which, at the same time, is a target for protein kinase C-dependent phosphorylation. We show that Ca2+-dependent calmodulin binding to CaT-L, which facilitates channel inactivation, can be counteracted by protein kinase C-mediated phosphorylation of the calmodulin binding site.

Calmodulin is a selective mediator of Ca(2+)-induced Ca2+ release via the ryanodine receptor-like Ca2+ channel triggered by cyclic ADP-ribose.

The ryanodine receptor-like Ca2+ channel (RyRLC) is responsible for Ca2+ wave propagation and Ca2+ oscillations in certain nonmuscle cells by a Ca(2+)-induced Ca2+ release (CICR) mechanism. Cyclic ADP-ribose (cADPR), an enzymatic product derived from NAD+, is the only known endogenous metabolite that acts as an agonist on the RyRLC. However, the mode of action of cADPR is not clear. We have identified calmodulin as a functional mediator of cADPR-triggered CICR through the RyRLC in sea urchin eggs. cADPR-induced Ca2+ release consisted of two phases, an initial rapid release phase and a subsequent slower release. The second phase was selectively potentiated by calmodulin which, in turn, was activated by Ca2+ released during the initial phase. Caffeine enhanced the action of calmodulin. Calmodulin did not play a role in inositol 1,4,5-trisphosphate-induced Ca2+ release. These findings offer insights into the multiple pathways that regulate intracellular Ca2+ signaling.

P2Y1 receptor-evoked glutamate exocytosis from astrocytes: control by tumor necrosis factor-alpha and prostaglandins.

ATP, released by both neurons and glia, is an important mediator of brain intercellular communication. We find that selective activation of purinergic P2Y1 receptors (P2Y1R) in cultured astrocytes triggers glutamate release. By total internal fluorescence reflection imaging of fluorescence-labeled glutamatergic vesicles, we document that such release occurs by regulated exocytosis. The stimulus-secretion coupling mechanism involves Ca2+ release from internal stores and is controlled by additional transductive events mediated by tumor necrosis factor-alpha (TNFalpha) and prostaglandins (PG). P2Y1R activation induces release of both TNFalpha and PGE2 and blocking either one significantly reduces glutamate release. Accordingly, astrocytes from TNFalpha-deficient (TNF(-/-)) or TNF type 1 receptor-deficient (TNFR1(-/-)) mice display altered P2Y1R-dependent Ca2+ signaling and deficient glutamate release. In mixed hippocampal cultures, the P2Y1R-evoked process occurs in astrocytes but not in neurons or microglia. P2Y1R stimulation induces Ca2+ -dependent glutamate release also from acute hippocampal slices. The process in situ displays characteristics resembling those in cultured astrocytes and is distinctly different from synaptic glutamate release evoked by high K+ stimulation as follows: (a) it is sensitive to cyclooxygenase inhibitors; (b) it is deficient in preparations from TNF(-/-) and TNFR1(-/-) mice; and (c) it is inhibited by the exocytosis blocker bafilomycin A1 with a different time course. No glutamate release is evoked by P2Y1R-dependent stimulation of hippocampal synaptosomes. Taken together, our data identify the coupling of purinergic P2Y1R to glutamate exocytosis and its peculiar TNFalpha- and PG-dependent control, and we strongly suggest that this cascade operates selectively in astrocytes. The identified pathway may play physiological roles in glial-glial and glial-neuronal communication.

Integrin-Specific Mechanoresponses to Compression and Extension Probed by Cylindrical Flat-Ended AFM Tips in Lung Cells

Cells from lung and other tissues are subjected to forces of opposing directions that are largely transmitted through integrin-mediated adhesions. How cells respond to force bidirectionality remains ill defined. To address this question, we nanofabricated flat-ended cylindrical Atomic Force Microscopy (AFM) tips with ~1 ��m2 cross-section area. Tips were uncoated or coated with either integrin-specific (RGD) or non-specific (RGE/BSA) molecules, brought into contact with lung epithelial cells or fibroblasts for 30 s to form focal adhesion precursors, and used to probe cell resistance to deformation in compression and extension. We found that cell resistance to compression was globally higher than to extension regardless of the tip coating. In contrast, both tip-cell adhesion strength and resistance to compression and extension were the highest when probed at integrin-specific adhesions. These integrin-specific mechanoresponses required an intact actin cytoskeleton, and were dependent on tyrosine phosphatases and Ca2+ signaling. Cell asymmetric mechanoresponse to compression and extension remained after 5 minutes of tip-cell adhesion, revealing that asymmetric resistance to force directionality is an intrinsic property of lung cells, as in most soft tissues. Our findings provide new insights on how lung cells probe the mechanochemical properties of the microenvironment, an important process for migration, repair and tissue homeostasis.